The objective of this project is to elucidate the regulation mechanism of bacterial chemotaxis. Based on the principle of universality among living creatures at the molecular level, an understanding of this mechanism in a simple organism such as Escherichia coli may have relevance to the sensory transduction mechanism in higher organisms and humans. Two approaches are used in this proposal. (A) I have developed from E. coli a sub-cellular system of cell envelopes, free of cytoplasm, to which flagella are still attached and capable of movement. Using this system, in which content can be controlled and changed at will, I shall try to reveal the nature of the chemical process that drives the flagella, and how the direction of flagellar rotation is controlled. (The direction of rotation of the flagella is central to the expression of bacterial chemotaxis.) This will be done by determining which ions are required for flagellar rotation and what ion fluxes occur, and by building chemical gradients of these ions to artificially restore flagellar rotation. Using this system, smooth mutants (primarily counterclockwise rotation) and tumbly mutants (primarily clockwise rotation) will be compared, followed by imitating the effect of stimuli, i.e. affecting and controlling the direction of flagellar rotation, still in this sub-cellular system. (B) The involvement of ions and changes in membrane potential in chemotaxis is controversial. My preliminary studies demonstrate, however, that if masking processes are blocked, stimulus-induced changes in membrane potential can be detected in intact bacteria, indicative of a role for ions in the chemotaxis machinery. Applying such conditions to intact E. coli cells, from a variety of mutants defective in different parts of the chemotaxis machinery, and using ion-selective electrodes or radioactive-labeled ions to follow transport processes, the following questions can be answered: Which ions are involved in chemotaxis? Where do they act? How do they act? If ions are indeed involved in chemotaxis, this approach ought to reveal the mechanism(s) of excitation and/or regulation in bacterial chemotaxis.